US9540420B2 - Mucosal vaccines - Google Patents

Mucosal vaccines Download PDF

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US9540420B2
US9540420B2 US13/582,160 US201113582160A US9540420B2 US 9540420 B2 US9540420 B2 US 9540420B2 US 201113582160 A US201113582160 A US 201113582160A US 9540420 B2 US9540420 B2 US 9540420B2
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antigen
mucosal
vaccine
vehicle
cvp
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US20130045232A1 (en
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Hiroshi Kido
Dai Mizuno
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University of Tokushima NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a mucosal vaccine inducing a mucosal IgA and a blood IgG effectively.
  • Patent Documents 1 and 2 made detailed descriptions of the demerits in conventional inactivated vaccines or toxoids, as well as the current states with regard to the development of mucosal vaccines and immunoadjuvants.
  • an antigen-drug (AD) vehicle which is a complex of a pulmonary surfactant protein B and/or a pulmonary surfactant protein C and a lipid(s), and a mucosal vaccine consisting of this AD vehicle and an antigen (Patent Document 1).
  • the present inventors also found that by adjusting the weight ratio V/A of the AD vehicle amount (V) to the antigen amount (A), the selective production of an IgA antibody and the production of both IgA and IgG antibodies are convertible, and then developed a mucosal vaccine based on such action mechanism (Patent Document 2).
  • Patent Documents 1 and 2 also disclose the effectiveness of fragments (peptides) of the pulmonary surfactant proteins B and C.
  • an AD vehicle comprising as a component a synthetic peptide KnLm (wherein n is 4 to 8 and m is 11 to 20) which, in spite that it is a smaller-sized peptide than the partial peptides disclosed in Patent Documents 1 and 2, has a potent antibody production-inducing or -enhancing effect, especially for an exclusive production of a secretory IgA antibody as well as an excellent and effective inductory effect on the production of both secretory IgA and blood IgG, and a mucosal vaccine consisting of this AD vehicle and an antigen (Patent Document 3).
  • a carboxyvinyl polymer (CVP) or a hydroxypropyl cellulose (HPC) is employed widely and a thickening gelator such as sodium alginate is also employed.
  • CVP carboxyvinyl polymer
  • HPC hydroxypropyl cellulose
  • a thickening gelator such as sodium alginate
  • an HPC-containing mucosal vaccine Patent Document 4
  • a CVP-containing mucosal vaccine formulation Patent Document 5
  • an influenza vaccine for nasal spray Patent Document 6
  • Patent Document 7 also discloses a mucosal vaccine consisting of an antigen, an adjuvant [especially Poly(I:C)] and a thickening agent (sodium alginate and the like).
  • the mucosal vaccine of Patent Document 3 has a potent antibody producing ability, it has a drawback, in common with other mucosal vaccines, that it requires an antigen in an amount larger than that in a percutaneously injectable vaccine.
  • a vaccine in an amount sufficient for the prevalence areas of its target infection should be required, but in view of the current antigen production scale it is difficult to produce an increased amount of the mucosal vaccine. Accordingly, it is desired to impart a further potent antibody producing ability to the mucosal vaccine.
  • An object of the invention is to provide an improved mucosal vaccine having a more potent ability in antibody producing than those of mucosal vaccines described in Patent Documents 3, and, as a result, being capable of exerting an excellent effect comparable to a subcutaneously injectable vaccine, even with an extremely small amount of an antigen.
  • the inventors as a means for further enhancing the antibody induction ability of the mucosal vaccines of Patent Documents 3, examined gelators (CVP, HPC) employed in a nasal drop or a mucosally applicable vaccine whether they can function in AD vehicle to increase the amount of the antigen to be delivered to an antigen presenting cell by nasal-clearance prolongation and thereby inducing a mucosal immune IgA and a blood immune IgG, and the followings were confirmed.
  • CVP gelators
  • the invention was established based on the novel findings above.
  • the invention is a mucosal vaccine producing an antigen-specific mucosal IgA and blood IgG in the levels capable of exerting an effective immune induction and an infection-preventing effect, which comprises:
  • the antigen protein (c) is in an amount, even in combination with the AD vehicle (a) or in combination with the carboxyvinyl polymer (b), incapable of producing an antigen-specific mucosal IgA and blood IgG sufficient for exerting an effective immune induction and an infection-preventing effect.
  • the synthetic peptide consists of the amino acid sequence of SEQ ID NO: 1 or 2.
  • the lipid is at least one of phosphatidyl choline, dipalmitoylphosphatidyl choline, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidic acid, lauric acid, myristic acid, palmitic acid, stearic acid and oleic acid. More specifically, the lipids are a mixture of dipalmitoylphosphatidyl choline, phosphatidyl glycerol and palmitic acid.
  • the antigen is a pathogen-derived inactivated antigen, a purified antigen, a partially purified antigen, a recombinant antigen, a detoxicated toxin, or an allergen causative of an allergy.
  • the present invention is also a method for producing a mucosal vaccine producing an antigen-specific mucosal IgA and blood IgG in the levels capable of exerting an effective immune induction and an infection-preventing effect, which comprises:
  • the phrase “amounts of a mucosal IgA and a blood IgG capable of exerting an effective immune induction” mans any amounts of IgA and IgG such that give the hemagglutination inhibition (HI) values of blood not lower than the international evaluation criteria.
  • composition consisting of a synthetic peptide and lipids may be referred to as an “AD vehicle”, and a composition consisting of the AD vehicle and an antigen may be referred to as a “mucosal vaccine”.
  • AD vehicle and “mucosal vaccine” are identical substantially to those disclosed in Patent Document 3.
  • a composition of the mucosal vaccine combined with a CVP may be referred to as a “CVP-added mucosal vaccine”.
  • the “lipids” encompasses the disclosures of Patent Documents 1 to 3
  • the “synthetic peptide” and the “AD vehicle” encompasses the disclosure of Patent Document 3.
  • the CVP-added mucosal vaccine of the present invention has an extremely high vaccine antigen-specific IgA and IgG antibody inducing effect and a potent hemagglutination inhibition (HI) effect of the induced antibody, which far exceeds the international protective standard.
  • HI hemagglutination inhibition
  • the CVP-added mucosal vaccine of the invention can exert a far potent antibody inducing effect even when the amount of the antigen employed is reduced to 1 ⁇ 5 or less then the amount in a conventional mucosal vaccine (antigen+synthetic peptide+lipids).
  • HI hemagglutination inhibition
  • composition itself (AD vehicle, CVP) of the CVP-added mucosal vaccine of the invention has no effect for stimulating antigen recognizing cells, and accordingly it has an extremely low possibility of developing an unexpected side effect such as autoimmune diseases or post-vaccination allergy exacerbation due to any antigens other than the vaccine antigen.
  • a mucosal vaccine having a further higher ability of producing the antigen-specific mucosal IgA and blood IgG can be produced.
  • FIG. 1 The results of Experiment 1, which indicate the nasal wash IgA levels (left) and serum IgG levels (right) when mice are treated nasally with either of the vaccines of Example 1 and Comparative Examples 1 to 5 or an HA antigen.
  • FIG. 2 The results of Experiment 1, which indicate the HI values exhibited by an anti-influenza virus HA antibody when mice are treated nasally with either of the vaccines of Example 1 and Comparative Examples 1 and 5, HA antigen or HA+AD vehicle as a reference control.
  • FIG. 3 The results of Experiment 2, which indicate the change in expression levels of activation marker molecules (MHC II, CD40, CD80(B7-1) and CD86(B7-2)) on the cell membrane, when antigen-presenting dendritic cells are stimulated with endotoxin (LPS), poly(I:C) and SF-10 (CVP+AD vehicle) respectively.
  • the left graph shows the ratio (%) of the positive cells in the total cell count, which indicates an increased expression of each membrane molecule (the cells show over the negative cut off value limit as indicating bar near the center, which was determined based on the saline treatment employed as a control in the dot graphs on the right).
  • the right shows the results of flow cytometry measurement, in which the amount of the activation marker molecules on the cell membrane is visualized.
  • FIG. 4 The results of Experiment 4, which indicate the relationship between the PFU amount of the infected influenza virus and the survival rate.
  • FIG. 5 The results of Experiment 4, which are the changes in the % survival when the mice (10 mice per group) were immunized intranasally with either of the vaccines of Example 1, Example 5, Comparative Example 5 or Comparative Example 6, and then infected with 50 PFU of the influenza virus.
  • FIG. 6 The results of Experiment 4, which are the changes in the % survival when the mice (10 mice per group) immunized intranasally with either of the vaccine of Example 1, Example 5, Comparative Example 5 or Comparative Example 6, and then infected with 800 PFU of the influenza virus.
  • FIG. 7 Transmission electron microscope images, the results of Experiment 6.
  • A The mucosal vaccine of Comparative Example 1
  • B CVP-added mucosal vaccine of Example 1 and the partially magnified image thereof (right figure).
  • FIG. 9 The result of Experiment 8, in which the expression levels of activation marker molecule (CD86) on the cell membrane were measured when antigen presenting dendritic cells were stimulated with poly(I:C) and SF-10 (CVP+AD vehicle), respectively.
  • the ⁇ indicates the absence of HA antigenic protein, while the ⁇ indicates the presence of HA antigenic protein.
  • MFI Mean Fluorescence Intensity
  • FIG. 10 The results of Experiment 9, in which CD86 expression levels on the membrane of dendritic cells prepared from a nasal cavity tissue of mice was measured. Mice were immunized intranasally with the SF-10 (CVP+AD vehicle) adjuvant in the presence or absence of the antigen. MFI: Mean Fluorescence Intensity.
  • the CVP-added mucosal vaccine of the invention consists of the following composition.
  • a synthetic peptide consists of the amino acid sequence of KnLm (wherein n is 4 to 8, and m is 11 to 20).
  • KnLm has n ⁇ K(Lys) residues on the N-terminus side and m ⁇ L residues on the C-terminus side.
  • Such a synthetic peptide may be any of the following peptides. In the parenthesis, the abbreviation of a peptide is indicated. The amino acid residue is indicated as a single letter code.
  • SEQ ID NO: 1 (K6L16) consists of 6 K(Lys) residues on the N-terminus side and 16 L residues on the C-terminus side
  • SEQ ID NO: 2 (K6L11) consists of 6 K(Lys) residues on the N-terminus side and 11 L residues on the C-terminus side.
  • These synthetic peptides should be those prepared according to known chemical synthesis methods whose purities are 95% or higher.
  • a phospholipid contained in a pulmonary surfactant such as phosphatidyl choline, dipalmitoylphosphatidyl choline, phosphatidyl serine and phosphatidyl glycerol is employed preferably. Otherwise, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidic acid, sphingomyelin, and the like may also be employed. As fatty acids, lauric acid, myristic acid, palmitic acid, stearic acid, palmitooleic acid, oleic acid, and the like may be employed. It is also possible to employ a lipid derived from aquatic animals such as whale and dolphin whose lungs are inflated dynamically.
  • the CVP is a hydrophilic polymer obtained by polymerizing acrylic acid as a main component, and commercially available ones such as Hivis Wako 103, Hivis Wako 104, Hivis Wako 105, Sigma Corporation's product pAA130 (Sigma, St. Louis, Mo., Cat No. 181293), pAA450 (Sigma, Cat No.181285) and pAA1250 (Sigma, Cat No.306215) can be employed. Among these, Hivis Wako 104, and Sigma Corporation's products pAA130 and pAA1250 which are employed widely in producing cosmetic and pharmaceutical gels are preferred.
  • a NaOH neutralizing solution can be employed to adjust to pH5.0 to 10.5, while it is preferred to employ a pH by which the stability of the vaccine antigen is not affected adversely.
  • an influenza split vaccine antigen is adjusted to pH6.8 to 8.0, preferably pH7.0 to 7.2.
  • the antigen includes antigen molecules such as a highly purified bacterial antigen for vaccination whose purity is about 90% or higher, a viral antigen, a protein such as a toxoid, a glycoprotein, an allergen, a polymeric saccharide and a nucleic acid.
  • antigen molecules such as a highly purified bacterial antigen for vaccination whose purity is about 90% or higher, a viral antigen, a protein such as a toxoid, a glycoprotein, an allergen, a polymeric saccharide and a nucleic acid.
  • the antigen for a vaccine against a chickenpox virus, a measles virus, a mumps virus, a poliovirus, a rotavirus, an influenza virus, an adenovirus, a herpes virus, a severe acute respiratory infection syndrome (SARS) virus, a West Nile virus, a Hantaan virus, a dengue virus, a Japanese encephalitis virus, a yellow fever virus, a tick-borne encephalitis virus, an HIV virus, a hepatitis C virus, a Bordetella pertussis , a meningococcus, an influenza B, a Pneumovirus, a Vibriocholera , a Plasmodium , a sleeping sickness pathogen and the like is contemplated.
  • the antigen is employed in the amount, when used alone, or especially when combined with AD vehicle (a) or combined with a carboxyvinyl polymer (CVP), which is adjusted so that the antigen-specific mucosal IgA and blood IgG in an amount producing an effective immune induction is not produced.
  • AD vehicle a
  • CVP carboxyvinyl polymer
  • the influenza antigenic protein contains a protein M, a neuraminase, a nucleoprotein and the like in addition to HA antigen molecule.
  • the antigenic protein amount means a total protein amount including the antigenic molecules listed above.
  • the amount of HA antigen molecule itself was about 50% of the total antigenic protein in the case of the influenza vaccine of the lot employed.
  • lipids from those listed above are mixed in a suitable ratio and suspended in a chloroform: methanol (2:1 (v/v)) mixture for example at a concentration as a lipid of 10 mg/mL, and employed as a lipid component.
  • the synthetic peptide is dissolved in ethanol for example at a concentration of 5.0 mg/mL. Then these lipid component and synthetic peptide are mixed.
  • the mixing ratio involves about 0.2 to about 12.0% by dry weight for the synthetic peptide, and about 88 to about 99.8% by dry weight for the lipid. This mixture is evaporated into dryness at about 40° C.
  • the aforementioned AD vehicle, CVP and antigen are mixed in a suitable ratio.
  • the AD vehicle solution is admixed in the vaccine stock solution so that the ratio of the AD vehicle amount (V) to the antigenic protein (A) on the dry weight basis, i.e. V/A, becomes a desired value.
  • the dry weight of the antigenic protein (A) to be administered to a single mouse is about 0.01 to about 10 pg/kg body weight, preferably about 0.03 to about 5.0 ⁇ g/kg body weight.
  • This antigenic protein amount is 1 ⁇ 5 or less of the antigen amount (about 0.1 to about 50 ⁇ g/kg body weight, preferably about 0.3 to about 30 ⁇ g/kg body weight) in the mucosal vaccine of Patent Document 3 (antigen+AD vehicle).
  • the V/A for inducing the IgA antibody production predominantly and selectively is preferably about 0.1 to about 1.0.
  • the V/A for inducing the production of both of the IgA and IgG antibodies is about 1.0 to about 100, preferably about 5 to about 20.
  • about 60% or more of the antigen is bound to the AD vehicle, and the resultant mucosal immune vaccine is capable of inducing the IgA antibody production and/or the IgG antibody production efficiently.
  • the CVP concentration of the final nasal vaccine solution to which CVP was added is about 0.1% to 1.0%, preferably 0.3% to 0.8%.
  • the AD vehicle, the antigen and CVP can be mixed uniformly using a homogenizer, a mixer, an agitator, a stirrer and the like.
  • Example 1 the antigenic protein and the AD vehicle are mixed, and then subjected to an ultrasonic treatment for 3 minutes, and then vortexed for 2 hours at room temperature, and finally combined with an equal volume of a 1% CVP solution in physiological saline, thereby producing the CVP-added mucosal vaccine.
  • This method is identical to the method described in Patent Document 3 except for adding CVP. Nevertheless, this method is disadvantageous since it may allow the viral antigen to become inactivated due to the heat generated by the ultrasonic treatment and the ultrasonic treatment process poses a difficulty in keeping a constant condition in view of the type of the instrument employed, the size of the oscillator, the state of the oscillator and the like.
  • Example 7 a production method exemplified in Example 7 is preferred, which method comprises the following steps:
  • the CVP-added mucosal vaccine thus prepared may be used in a single dosing, but it is used preferably in two dosings (initial immunization and secondary immunization) or three dosings (initial immunization, secondary immunization and tertiary immunization).
  • Such a repeated immunizing treatment allows the antibody titres of antigen-specific IgA and IgG to be increased markedly.
  • the two or three vaccine dosings are conducted at intervals of 1 week to 3 weeks, preferably about 2 weeks.
  • the administration of the CVP-added mucosal vaccine of the invention can be done to the nasal cavity as well as the oral cavity or the vaginal cavity (see for example Lubrizol Pharmaceutical Bulletin, Polymers for Pharmaceutical Applications, Lubrizol Advanced Materials, Inc. 2008).
  • An AD vehicle was prepared as described below. Dipalmitoylphosphatidylcholine (DPPC), phosphatidyl glycerol (PG) and palmitic acid (PA) were mixed in a ratio of 75:25:10 (w/w/w) and suspended in a chloroform:methanol (2:1 (v/v)) mixture solution at a concentration as a phospholipid of 10 mg/ml to obtain a lipid component.
  • a synthetic peptide K6L16 (KKKKKKLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL having a purity of 95% or higher was dissolved in methanol at 5.0 mg/mL.
  • the freeze dried AD vehicle was used as being suspended in physiological saline just before use.
  • the influenza vaccine (HA) antigen employed was A/NewCaledonia/20/99(H1N1) provided by The Research Foundation for Microbial Diseases of Osaka University at 1.94 mg protein/mL (sodium phosphate buffer solution, sodium chloride, thimerosal solution: containing, per ml, 3.53 mg of sodium hydrogen phosphate hydrate, 0.54 mg of sodium dihydrogen phosphate, 8.50 mg of sodium chloride and 0.008 mg of thimerosal).
  • the vaccine and the AD vehicle were mixed in such a ratio that the ratio of the antigen solution protein amount (A) to the AD vehicle solution phospholipid amount (V), i.e.
  • VA 10, subjected to an ultrasonic treatment involving On and OFF three times repetitively at intervals of 30 seconds to accomplish the ultrasonic treatment for 3 minutes in total including ON for 90 seconds and OFF for 90 seconds in total (model S-250D, Branson Ultrasonics Danbury), then vortexed for 2 hours at room temperature and dissolved in physiological saline, and then combined with a neutralized 1% CVP (Hivis Wako 104) at a final concentration of 0.5%.
  • a neutralized 1% CVP Hivis Wako 104
  • the pH of this CVP-added mucosal vaccine was within the range for allowing the antigenicity of the HA antigenic protein to be maintained (7.0 to 7.2).
  • the SF-10 amounts in other Examples are indicated as values excluding the CVP amounts.
  • Example 1 According to the method in Example 1, a CVP-added mucosal vaccine of which HA antigenic protein amount was 0.1 ⁇ g was prepared.
  • the mixing ratio of the HA antigenic protein, the AD vehicle and CVP was identical to that in Example 1 (i.e., containing the AD vehicle in an amount 10 times the antigenic protein amount and CVP at a final concentration of 0.5%).
  • Example 1 According to the method in Example 1, a CVP-added mucosal vaccine of which HA antigenic protein amount was 0.03 ⁇ g was prepared.
  • the mixing ratio of the HA antigenic protein, the AD vehicle and CVP was identical to that in Example 1 (i.e., containing the AD vehicle in an amount 10 times the antigenic protein amount and CVP at a final concentration of 0.5%).
  • the mucosal vaccine of Patent Document 3 (HA+AD vehicle) was prepared.
  • the HA antigenic protein and the AD vehicle were identical to those in Example 1, and the preparation of the vaccine was conducted as same to Example 1.
  • the HA antigenic protein amount was 0.2 ⁇ g and the AD vehicle amount was 2.0 ⁇ g.
  • the mucosal vaccine disclosed in Patent Documents 5 and 6 was prepared.
  • the HA antigenic protein and CVP were identical to those in Example 1, and the preparation of the vaccine was conducted as same to Example 1.
  • the HA antigenic protein amount was 0.2 ⁇ g and the final concentration of CVP was 0.5%
  • the mucosal vaccine disclosed in Patent Document 4 was prepared.
  • the HA antigenic protein was identical to that in Example 1, and HPC was a commercially available HPC 6.0-10.0 (Wako Pure Chemical Industries, Ltd.)
  • the preparation of the vaccine was conducted as same to Example 1.
  • the HA antigenic protein amount was 0.2 ⁇ g and the HPC amount was 20 ⁇ g.
  • a mucosal vaccine (HA+AD vehicle+HPC) was prepared by combining the HA+AD vehicle of Patent Documents 3 with HPC.
  • the HA+AD vehicle was identical to that in Comparative Example 1 and HPC was identical to that in Comparative Example 3, while the preparation of the vaccine was conducted as same to Example 1.
  • the HA antigenic protein amount was 0.2 ⁇ g
  • the AD vehicle amount was 2.0 ⁇ g
  • the HPC amount was 20 ⁇ g.
  • a mucosal vaccine (HA+poly (I:C)) containing a poly (I:C) (Alexis Corp.) which is a ligand of a Toll-Like Receptor (TLR) of a dendritic cell and which stimulates an antigen presenting cell strongly to promote the antibody production was prepared in accordance with the method described in a publication (Asahi-Ozaki Y et al., Microbes Infect 2006; 8:2706-2714, Ichinohe T, et al., J Virol 2005; 79(5): 2910-2919).
  • the HA antigenic protein amount is 0.2 ⁇ g and the poly (I:C) amount is 2 ⁇ g.
  • the HA antigen was diluted with physiological saline and employed as a vaccine.
  • the HA antigenic protein dose per animal was 0.2 ⁇ g.
  • mice Using mice, the antibody production enhancing effects of the nasal mucosal vaccines were tested.
  • mice Female BALB/c mice (6-8 week-old) purchased from Japan SLC, Inc. (Shizuoka, Japan) were used. All animal experiments were conducted in an infected animal building (level P2) in Institute for Animal Experimentation, The University of Tokushima, School of Medicine, in accordance with the guidelines of Committee for Animal Experimentation of University of Tokushima, School of Medicine.
  • the secondary boost immunization was conducted by the nasal administration of the identical dose of vaccines at two weeks after the initial immunization. After the secondary immunization for two weeks, the tertiary immunization was conducted by a similar method, and at two weeks after last immunization the samples were taken. While the vaccine was given three times in total, the secondary immunization as the final immunization can give almost similar results.
  • a vaccine-treated mouse was anesthetized with pentobarbital to a thoracolaparotomy, and the tracheal duct was incised to insert an Atom venous catheter 3 Fr having nodes (Atom Medical Corporation, Tokyo, Japan) into the lung, to which then 1 mL of physiological saline was infused and then recovered. This procedure was repeated three times and the resultant 3 mL in total was employed as an alveolar wash. After collecting the alveolar wash, an Atom venous catheter was inserted via the incised tracheal duct to the direction of the nasal cavity, to which 1 mL of the physiological saline was infused and the fluids coming out of the nose were collected. The fluids were employed as nasal washes. In addition, a blood was taken out from the heart, centrifuged for 10 minutes at 5000 rpm to prepare a serum.
  • ELISA assay was conducted according to the method for a Mouse ELISA quantitation kit of BETHYL LABORATORIES, INC. (Texas, United States). To each well of a 96-well Nunc immunoplate (Nalgen Nunc International, New York, United States), 1 ⁇ g of the vaccine and 100 ⁇ L of a 1 ⁇ g/mL PBS solution of bovine serum albumin (BSA, SIGMA, Missouri, United States) were added, and allowed to undergo a solid phase immobilization reaction overnight at 4° C. Thereafter, the vaccine fluid was removed by washing three times with a washing fluid (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0).
  • BSA bovine serum albumin
  • a goat anti-mouse IgA or IgG-horse radish peroxidase (HRP) (BETHYL LABORATORIES INC.) was employed as a secondary antibody, and a TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Inc. Maryland, United States) was employed to conduct a chromogenic reaction.
  • the reaction was terminated by adding 100 ⁇ L of a 2 M H 2 SO 4 (Wako Pure Chemical Industries, Ltd.) to each well, and the absorbance at 450 nm was measured using SPECTRAmax PLUS 384.
  • the absorbance obtained similarly using 10 ng of the anti-influenza IgA and IgG purified from the aforementioned pulmonary washing was employed.
  • FIG. 1 The results of the anti-influenza HA antibody induction are shown for the IgA in the nasal washes ( FIG. 1 , left) and for the IgG in the blood ( FIG. 1 , right).
  • the respective measured values are shown in Table 1.
  • FIG. 2 and Table 2 show the HI titers of the respective test groups.
  • a antigen presenting dendritic cells prepared by the method of publication was stimulated with each of an endotoxin (LPS: 100 ng/1 ⁇ 10 5 cells) (Grauer O, et al., Histochem Cell Biol 2002; 117:351-362), poly(I:C) (10 ⁇ g/1 ⁇ 10 5 cells) and SF-10 (CVP-added AD vehicle 10 ⁇ g/1 ⁇ 10 5 cells), and the expression levels of the cell membrane activation marker molecules (MHC II, CD40, CD80(B7-1) and CD86(B7-2)) were measured.
  • LPS endotoxin
  • poly(I:C) 10 ⁇ g/1 ⁇ 10 5 cells
  • SF-10 CVP-added AD vehicle 10 ⁇ g/1 ⁇ 10 5 cells
  • LPS and poly (I:C) increased the expression of CD40 and CD86 markedly and activated the antigen presenting dendritic cells, while SF-10 itself did not increase the expression of MHC II, CD40, CD80 and CD86 on the antigen presenting dendritic cells, showing a level almost equal to the expression in the control (non-treatment cell).
  • Example 1 HA antigenic protein amount: 0.2 ⁇ g
  • Example 2 HA antigenic protein amount: 0.1 ⁇ g
  • Example 3 HA antigenic protein amount: 0.03 ⁇ g
  • Example 1 HA antigenic protein amount: 0.2 ⁇ g
  • Example 2 HA antigenic protein amount: 0.1 ⁇ g
  • Example 3 HA antigenic protein amount: 0.03 ⁇ g
  • Comparative Example 2 HA+AD vehicle (HA antigenic protein amount: 0.2 ⁇ g)
  • Comparative Example 3 HA+CVP (HA antigenic protein amount: 0.2 ⁇ g) in Experiment 1 (Table 1).
  • Example 3 treatment group exhibited an antigen-specific IgA level which was 11.7 times of that in Comparative Example 2 and 8.0 times of that in Comparative Example 3, and an IgG amount which was 77.2 times of that in Comparative Example 2 and 60.1 times of that in Comparative Example 3.
  • Example 1 a CVP-added mucosal vaccine having an AD vehicle amount of 0.3 ⁇ g and an HA antigenic protein amount of 0.03 ⁇ g was prepared.
  • the mixing ratio of the HA antigenic protein, the AD vehicle and CVP was identical to that in Example 1 (i.e., containing the AD vehicle in an amount 10 times the antigenic protein amount and CVP at a final concentration of 0.5%).
  • Example 1 Each CVP-added mucosal vaccine of Example 1 (AD vehicle: 2.0 ⁇ g, HA antigenic protein amount: 0.2 ⁇ g) and Example 4 (AD vehicle: 0.3 ⁇ g, HA antigenic protein amount: 0.03 ⁇ g) was administered to mice (10 animals in each group) similarly to Experiment 1, which was then infected at day 14 after the second booster vaccination (immunization 3 times in total) with 50 PFU and 800 PFU of an influenza virus (A/PR8(N1H1)/ ⁇ L).
  • the infecting viral amount capable of obtaining a 50% lethal dose (LD50) was PFU ⁇ 5, and in the absence of the vaccine treatment all mice died within 9 days at 50 PFU and 8 days at 100 PFU or more after the infection.
  • mice treated with the CVP-added mucosal vaccines of Example 1 and Example 4 all survived for 15 days after viral infection with 50 PFU.
  • the viral infection with 800 PFU killed 1 out of 10 animals treated with the vaccine of Example 1 (after 8 days or later) and 5 out of 10 animals treated with the vaccine of Example 4 (after 10 days or later).
  • the CVP-added mucosal vaccine of the invention was confirmed to have an excellent infection preventing effect.
  • a freeze dried AD vehicle powder was dissolved in a pure water and added to the HA antigenic protein fluid similar to that in Example 1, and then this mixture solution was combined with an equal quantity of a 1.0% CVP dissolved in a pure water to prepare a suspension.
  • This suspension was warmed, without ultrasonic treatment, at 42° C. for 10 minutes using a water bath, and at the times of 3 and 7 minutes during the warming treatment the solution was stirred using a mixer for 10 seconds to achieve uniformity. After the warming treatment, the suspension was frozen overnight at ⁇ 30° C. to ⁇ 75° C., and lyophilized to make a dried powder. The freeze dried powder was stored at ⁇ 30° C.
  • the freeze dried powder was suspended in physiological saline to obtain a CVP-added mucosal vaccine.
  • the vaccine solution was adjusted so that 4 ⁇ l in total to be administered to the both nasal cavities of a mouse with 2 ⁇ l being given to each nostril a solution containing CVP at 0.5%, 0.2 ⁇ g of the HA antigenic protein and 2.0 ⁇ g of the AD vehicle phospholipid.
  • this CVP-added mucosal vaccine is designated as HA+SF-10-B.
  • a freeze dried AD vehicle powder dissolved in a pure water was admixed with the HA antigenic protein fluid similar to that in Example 1. This suspension was warmed at 42° C. for 10 minutes using a water bath, and at the times of 3 and 7 minutes during the warming treatment the solution was stirred using a mixer for 10 seconds to achieve uniformity. After the warming treatment, the suspension was frozen overnight at ⁇ 30° C. to ⁇ 75° C., and lyophilized to make a dried powder. The freeze dried powder was stored at ⁇ 30° C. Just before use, the freeze dried powder was stirred gently with a 0.5% CVP solution previously dissolved in physiological saline while avoiding any foaming to obtain a CVP-added mucosal vaccine.
  • HA+SF-10-C This CVP-added mucosal vaccine is designated as HA+SF-10-C.
  • HA+SF-10-A Each CVP-added mucosal vaccine of Example 1 (hereinafter designated as “HA+SF-10-A”), Example 5 (HA+SF-10-B), and Example 6 (HA+SF-10-C) was inoculated to mice (10 animals in each group) similarly to Experiment 1 (three times at intervals of 2 weeks), and the anti-influenza IgA and IgG antibodies were measured similarly to Experiment 1.
  • Example 6 (HA+SF-10-C) exhibited an antibody inducing effect about 2 to 4 times for the IgA level and about 2 times for the IgG level, when compared with Example 1 (HA+SF-10-A) and Example 5 (HA+SF-10-B).
  • the method of Example 6 (Manufacturing Process C) was assumed to result in its excellent antibody inducing effect because no ultrasonic treatment step is involved and the CVP solution is added in the final step.
  • each component in the CVP-added mucosal vaccines of the invention was analyzed by a transmission electron microscope. As shown in FIG. 7 , CVP played a role in a substantial increase in the binding between the antigen and the AD vehicle.
  • CVP has been employed as a thickening agent (Patent Documents 5 and 6) which is one of thickening polymers for improving the residence of an active ingredient in the nasal cavity in the same manner as hydroxypropyl cellulose (Patent Document 4), sodium alginate (Patent Document 7) and other excipients.
  • Patent Documents 5 and 6 which is one of thickening polymers for improving the residence of an active ingredient in the nasal cavity in the same manner as hydroxypropyl cellulose (Patent Document 4), sodium alginate (Patent Document 7) and other excipients.
  • CVP in the present invention was revealed, in a combination with the AD vehicle, to enhance the binding between the antigen and the AD vehicle, and to increase the amount of the antigen to be incorporated into the antigen presenting cells, thus enhancing the AD vehicle effect.
  • SF-10 (AD vehicle+CVP) (20 ⁇ g) of Example 1 and the HA antigenic protein (2 ⁇ g) of Example 1 which was labeled with a fluorescent dye ATTO 488 were mixed and then added to the mouse bone marrow-derived dendritic cells (2 ⁇ 10 5 cells), and after 1 hour incubation the fluorescence intensity of the dendritic cell labeled with the fluorescent dye-labeled HA antigenic protein was measured by a flow cytometry.
  • the poly (I:C) (10 ⁇ g) of Comparative Example 5 and the fluorescent-labeled HA antigenic protein (2 ⁇ g) were mixed and the fluorescence intensity of the dendritic cells was measured similarly.
  • the fluorescence exhibited by the dendritic cells reflects the binding and incorporation of the fluorescent dye-labeled HA antigenic protein to the cells.
  • the SF-10 mucosal vaccine of the invention was confirmed to promote the binding and the incorporation of the antigenic protein to the antigen presenting cells thereby increasing the antibody production and exerting an excellent infection preventing ability.
  • the followings were added to the cultured mouse bone marrow-derived dendritic cells (2 ⁇ 10 5 cells) in 1 mL of cRPMI medium, and the activation of the dendritic cells after 1 hour was measured by a flow cytometry employing as an index of the increase in the expression of CD86 which is one of the dendritic cell activation markers.
  • FACSCalibur cytometer (BD Biosciences, Massachusetts, United States) was used for measurement and CellQuest software (BD Biosciences, Massachusetts, United States) was used for data processing to determine the CD86 expression.
  • the HA antigenic protein (2 ⁇ g) and SF-10 (20 ⁇ g) were those described in Example 1, while Poly (I:C) (10 ⁇ g) was identical to that in Comparative Example 5.
  • the CVP-added mucosal adjuvant of the invention i.e., SF-10
  • SF-10 the CVP-added mucosal adjuvant of the invention
  • the animal experiment was along the line with Experiment 1.
  • the HA antigenic protein (0.2 ⁇ g) and SF-10 (2 ⁇ g) were those of Example 1, while Poly (I:C) (2 ⁇ g) was identical to that in Comparative Example 5.
  • the mouse was decapitated 48 hours after the nasal inoculation and the tissues in the nasal cavities were collected and treated with a collagenase (1 mg/mL, shaking at 37° C. for 30 minutes). After filtration through a mesh followed by centrifugation (4° C., 10 min, 200 ⁇ g), dendritic cells were prepared from the recovered cells by VarioMACS (Miltenyl Biotech, Bergisch Gladbach, Germany) using Anti-CD11c (N-418)-conjugated magnetic beads (Miltenyl Biotech, Bergisch Gladbach, Germany) and LS column, in accordance with the manufacture's instruction. Then, the expression enhancement of CD86 was measured by the method similar to that in Experiment 8.
  • the results are shown in FIG. 10 .
  • the dendritic cell-activation due to an increased antigen delivery by SF-10 was confirmed also in the nasal cavities of the mouse.
  • CVP was investigated for its preferable concentration. Except for employing pAA130 manufactured by Sigma Corporation as CVP at concentrations of 0.1%, 0.25%, 0.5%, 0.75% or 1.0%, a CVP-added mucosal vaccine was prepared by the method of Example 6, and the amounts of mouse nasal wash antigen-specific IgA and serum IgG were measured by the method of Experiment 1.

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